A novel Time-resolved Fluoroimmunoassay for the quantitative detection of Antibodies against the Phospholipase A2 Receptor.

A highly sensitive time-resolved fluoroimmunoassay (TRFIA) was developed to quantify serum antibodies against the phospholipase A2 receptor (anti-PLA2R-IgG) for differential diagnosis of membranous nephropathy. Recombinant PLA2R (rPLA2R) was coated onto 96-well plates as a capture. A goat-anti-human IgG tracer was prepared with europium-chelate for detection. After bound/free separation by washing, the fluorescence counts of bound tracer were measured for quantifying serum anti-PLA2R-IgG concentration. A purified anti-PLA2R-IgG calibrator was first prepared for ensuring that consistent quantitative results could be obtained. The assay detection limit was 0.03 mg/L with linear measurement range of 0.03-340 mg/L. The intra- and inter-assay coefficients of variation (CVs) were 3.8% and 6.2%, respectively. The average serum anti-PLA2R-IgG concentration in 45 healthy volunteers, 31 IgA nephropathy, 9 lupus nephropathy, and 52 idiopathic membranous nephropathy patients was 0.53 ± 0.18 mg/L, 0.70 ± 0.41 mg/L, 1.08 ± 0.65 mg/L, and 9.00 ± 11.82 mg/L, respectively. The cut-off point for an abnormal anti-PLA2R-IgG concentration was defined as >0.89 mg/L. The positive rates in serum from patients with IgA nephropathy, lupus nephropathy, and idiopathic membranous nephropathy were 29.0%, 44.4%, and 88.5%, respectively. The availability of this quantitation method will facilitate the use of serum anti-PLA2R-IgG for diagnosing idiopathic membranous nephropathy.

Serum Pepsinogen Levels Are Correlated With Age, Sex and the Level of Helicobacter pylori Infection in Healthy Individuals.

BACKGROUND: To explore the relationship between age, sex, the level of Helicobacter pylori (HP) infection and serum pepsinogen (PG) in healthy people undergoing a medical examination. METHODS: A total of 6,596 "healthy" individuals undergoing a medical examination were selected as subjects in this study. The concentrations of serum pepsinogen I (PGI) and serum pepsinogen II (PGII) were tested for each of the subjects using time-resolved fluorescence immunoassay characterized with high sensitivity and wide measuring range. The infection ratio and level of HP were tested using a 13C-urea breath test to analyze the relationship between age, sex, HP infection, and serum PGs. RESULTS: The PGI, PGII and PGI-to-PGII ratio (x¯±S) were higher in males than in females. The serum PGI and PGII levels gradually increased with age. HP infection rate was 48.83%, and the serum PGI, PGII and PGI-to-PGII ratio (x¯±S) were 187.05 ± 73.50µg/L, 18.09 ± 8.68µg/L and 11.67 ± 5.44, respectively in the HP-positive group and 150.39 ± 67.04µg/L, 11.50 ± 7.45µg/L and 15.67 ± 8.19, respectively in the HP-negative group. There was significant difference in the detection rate of an abnormal PG between the 2 groups as with the worsening of HP infection, 13C-urea breath test and serum PGI and PGII levels increased, but the PGI-to-PGII ratio decreased significantly. CONCLUSIONS: Serum PGI and PGII levels were correlated with age, sex and the level of HP infection. Therefore, the influencing factors of age, sex and the level of HP infection should be considered when screening stomach diseases using PG.

Simultaneous detection of different serum pepsinogens and its primary application.

AIM: To develop the simple, rapid and sensitive dual-label time-resolved fluoroimmunoassay for pepsinogens in human serum. METHODS: Based on two-site sandwich protocol, monoclonal antibodies (McAbs) against pepsinogen I (PG I) and PG II were co-coated in 96 microtitration wells, and tracer McAbs against PG I and PG II were labeled with europium (Eu) and samarium (Sm) chelate, respectively. Diluted serum samples of Eu(3+)- and Sm(3+)-McAbs were added into microtitration wells simultaneously. After washing, fluorescence of bound Sm(3+) and Eu(3+) tracers was detected. RESULTS: The detection limit was 0.2 microg/L for PG I and 0.05 microg/L for PG II. The assay range was 5.0-320.0 microg/L for PG I and 1.0-55.0 microg/L for PG II. The average recovery rate was 102.7% for PG I and 98.8% for PG II. Sera from healthy controls and patients with gastric disease were analyzed. The PG detected by dual-label assay was in good agreement with that detected by single-label assay or by enzyme-linked immunosorbent assay. CONCLUSION: Dual-label assay can provide high-throughput serological screening for gastric diseases.

Dual-label time-resolved fluoroimmunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A.

A dual-label time-resolved fluoroimmunoassay (TRFIA) for simultaneous quantification of aflatoxin B1 (AFB1) and ochratoxin A (OTA) is described. For this, microtitration wells were coated with AFB1-horse radish peroxidase (HRP) and OTA-bovine serum albumin. The standards and samples were loaded on the coated plates, and diluted antibodies and Eu3+- and Sm3+-labeled IgG were then added. Our results showed that the sensitivity of TRFIA for AFB1 was 0.02 microg/L (range 0.02-100 microg/L). The intra- and inter-batch coefficient of variation (CV) was 3.2 and 7.3%, respectively, and the average recovery rate was 88.1%. On the other hand, the sensitivity of OTA was 0.05 microg/L (range 0.05-50 microg/L), the intra- and inter-batch CV was 2.9 and 7.9%, respectively, and the average recovery rate was 89.9%. In the AFB1/OTA-TRFIA, AFB1 and OTA did not mutually interfere. The correlation coefficients between the dual-label AFB1/OTA-TRFIA and the single-label AFB1-TRFIA or OTA-TRFIA were 0.972 and 0.981, respectively, indicating that the results were consistent. Our study suggests that AFB1/OTA-TRFIA allows the simultaneous detection of AFB1 and OTA; is a simple, fast, and economic method for screening large quantities of samples, and has good prospects of application.

Ultrasensitive detection of pepsinogen I and pepsinogen II by a time-resolved fluoroimmunoassay and its preliminary clinical applications.

A fast and highly sensitive assay for pepsinogen I (PG I) and pepsinogen II (PG II) by using time-resolved fluoroimmunoassay (TRFIA) detection technique has been developed for the determination of serum PG I and PG II against gastrointestinal diseases. On the noncompetitive assay, one monoclonal antibody (McAb) coated on wells was directed against a specific antigenic site on the PG I or PG II. The McAb, called as labelling McAb, was prepared with the europium-chelate of N-(p-isothiocyanatobenzyl)-diethylenetriamine-N,N,N,N-tetraacetic acid and directed against a different antigenic site on the PG I or PG II molecule. After bound/free separation by washing, the fluorescence counts of bound Eu(3+)-McAb were measured. The levels of PG in sera from patients or healthy volunteers were determined by PG I and PG II TRFIA using the autoDELFIA(1235) system. The measurement ranges of PG I-TRFIA were 3.5-328.0 microg L(-1) and those of PG II-TRFIA were 2.0-55.0 microg L(-1). The within-run and between-run CVs of the PG I-TRFIA were 1.9% and 4.7%, respectively, and those of PG II-TRFIA were 2.1% and 3.8%, respectively. The recovery rates of PG I-TRFIA and PG II-TRFIA were 102.7% and 104.6%, respectively. The detection limitations of PG I and PG II were 0.05 microg L(-1) and 0.02 microg L(-1), respectively. The dilution experiments showed the percentage of expected value of PG I-TRFIA was 93.2-102.3% and of PG II-TRFIA was 97.3-110.6%. The cross-reacting rate between PG I and PG II was negligible. The linear correlation of radioimmunoassay (RIA) and TRFIA measurements resulted in a correlation coefficient as 0.926 of PG I and as 0.959 of PG II. The europium-labelling McAbs were stable for at least one year at -20 degrees C, and the results of the TRFIA with same reagents were reproducible over one year as well. The means of 1600 healthy volunteers were 162.4+/-52.1 microg L(-1) for serum PG I, 11.7+/-6.8 microg L(-1) for serum PG II, and 13.8+/-7.4 for the PG I/PG II ratio. The normal ranges of Serum PG I levels for healthy volunteers were 58.2-266.6 microg L(-1), and those of serum PG II levels were less than 25.3 microg L(-1). The availability of a highly sensitive, reliable, and convenient PG-TRFIA method for quantifying PG will allow investigations into the possible diagnostic value of this analysis in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastric ulcer and gastritis. The sensitivity and reproducibility of the assay were satisfactory for clinical applications.